Tertiary Structural Rearrangements upon Oxidation of Methionine¹⁴⁵ in Calmodulin Promotes Targeted Proteasomal Degradation

详细信息    查看全文

• 作者：Colette A. Sacksteder ; Jennifer E. Whittier ; Yijia Xiong ; Jinhui Li ; Nadezhda A. Galeva ; Michael E. Jacoby ; Samuel O. Purvine ; Todd D. Williams ; Martin C. Rechsteiner ; Diana J. Bigelow ; Thomas C. Squier
• 刊名：Biophysical Journal
• 出版年：2006
• 出版时间：15 August 2006
• 年：2006
• 卷：91
• 期：4
• 页码：1480-1493
• 全文大小：372 K

文摘

The selectivity underlying the recognition of oxidized calmodulin (CaM) by the 20S proteasome in complex with Hsp90 was identified using mass spectrometry. We find that degradation of oxidized CaM (CaM_ox) occurs in a multistep process, which involves an initial cleavage that releases a large N-terminal fragment (A¹-F⁹²) as well as multiple smaller carboxyl-terminus peptides ranging from 17 to 26 amino acids in length. These latter small peptides are enriched in methionine sulfoxides (Met(O)), suggesting a preferential degradation around Met(O) within the carboxyl-terminal domain. To confirm the specificity of CaM_ox degradation and to identify the structural signals underlying the preferential recognition and degradation by the proteasome/Hsp90, we have investigated how the oxidation of individual methionines affect the degradation of CaM using mutants in which all but selected methionines in CaM were substituted with leucines. Substitution of all methionines with leucines except Met¹⁴⁴ and Met¹⁴⁵ has no detectable effect on the structure of CaM, permitting a determination of how site-specific substitutions and the oxidation of Met¹⁴⁴ and Met¹⁴⁵ affects the recognition and degradation of CaM by the proteasome/Hsp90. Comparable rates of degradation are observed upon the selective oxidation of Met¹⁴⁴ and Met¹⁴⁵ in CaM-L7 relative to that observed upon oxidation of all nine methionines in wild-type CaM. Substitution of leucines for either Met¹⁴⁴ or Met¹⁴⁵ promotes a limited recognition and degradation by the proteasome that correlates with decreases in the helical content of CaM. The specific oxidation of Met¹⁴⁴ has little effect on rates of proteolytic degradation by the proteasome/Hsp90 or the structure of CaM. In contrast, the specific oxidation of Met¹⁴⁵ results in both large increases in the rate of degradation by the proteasome/Hsp90 and significant circular dichroic spectral shape changes that are indicative of changes in tertiary rather than secondary structure. Thus, tertiary structural changes resulting from the site-specific oxidation of a single methionine (i.e., Met¹⁴⁵) promote the degradation of CaM by the proteasome/Hsp90, suggesting a mechanism to regulate cellular metabolism through the targeted modulation of CaM abundance in response to oxidative stress.